Stable NO-delivering compounds

ABSTRACT

Disclosed are novel NO-releasing compounds which comprise a stabilized S-nitrosyl group and a free alcohol or a free thiol group. Also disclosed is a method of preparing the NO-releasing compounds. The method comprises reacting a polythiol or a thioalcohol with a nitrosylating agent. Also disclosed are medical devices coated with the disclosed compounds, methods of delivering NO to treatments sites in a subject by utilizing the medical devices and methods of sterilizing surfaces.

BACKGROUND OF THE INVENTION

1. Nitric oxide (referred to herein as “NO”) has many uses, including asa medicinal agent. For example, NO has been shown to inhibit smoothmuscle proliferation, thrombus formation, platelet aggregation, andsmooth muscle contraction. NO can also be used as a bacteriocidal orbacteriostatic agent to sterilize the surfaces of, for example, medicaldevices. However, the half-life for NO release of most of the knowncompounds which release NO is less than twelve hours. Thus, most knownNO-releasing compounds are too unstable to be useful commercially. Thefull commercial potential of NO is unlikely to be realized until morestable NO-releasing compounds are developed.

SUMMARY OF THE INVENTION

2. It has now been found that an S-nitrosyl group (an “—S—NO” group) ina compound generally is stabilized when the compound also has a freethiol group or a free alcohol group. For example, the half-life forNO-release from thiol or alcohol bearing S-nitrosylated compounds isgenerally greater than about two hundred hours when nitrosylated withbetween about 0.5 to about 0.7 equivalents ofS-nitroso-N-acetyl-D,L-penicillamine (SNAP) (Example 2). In contrast,the half-life for NO release from most S-nitrosylated compounds withoutfree thiols or alcohols is typically less than twelve hours. Based onthese results, novel compounds with stabilized S-nitrosylated groups andmethods of preparing these compounds are disclosed. Also disclosed aremedical devices coated with the disclosed compounds, methods ofdelivering NO to treatments sites in a subject by utilizing the medicaldevices and methods of sterilizing surfaces.

3. One embodiment of the present invention is an NO-releasing compoundcomprising an S-nitrosyl group and a free alcohol or a free thiol group.The S-nitrosyl group is stabilized with the alcohol or thiol group.

4. Another embodiment of the present invention is a compound prepared byreacting a polythiol or a thioalcohol with a nitrosylating agent.Preferably, from about 0.5 to about 0.7 equivalents of nitrosylatingagent for each free thiol and each free alcohol group in the polythiolor thioalcohol is used. A “polythiol” is a compound with at least twofree thiol groups. A “thioalcohol” is a compound with at least one freealcohol and at least one free thiol group.

5. Yet another embodiment of the present invention is a method ofpreparing an NO-releasing compound comprising at least one S-nitrosylgroup and at least one free alcohol or free thiol group, wherein theS-nitrosyl group is stabilized with the alcohol or thiol group. Themethod comprises reacting a polythiol or a thioalcohol with anitrosylating agent. Preferably, from about 0.5 to about 0.7 equivalentsof nitrosylating agent for each free thiol and each free alcohol groupin the polythiol or thioalcohol are used.

6. Another embodiment of the present invention is an article which iscapable of releasing NO. The article contains (e.g., incorporates or iscoated with) at least one of the compounds of the present invention. Thearticle can be a device for which a useful result can be achieved by NOrelease, including a medical device suitable for implantation at atreatment site in a subject. The medical device can deliver nitric oxideto the treatment site in the subject after implantation. In anotherexample, the article is, for example, a tube or catheter for contactinga bodily fluid of a subject.

7. Another embodiment of the present invention is a method of deliveringnitric oxide to a treatment site in a subject. A medical device whichcontains one or more of the compounds of the present invention isimplanted into the subject at the treatment site.

8. Another embodiment of the present invention is a method of deliveringnitric oxide to a bodily fluid of a subject. The method comprisescontacting the bodily fluid with an article, for example a tube orcatheter, which contains at least one of the compounds of the presentinvention.

9. Yet another embodiment of the present invention is a method ofinhibiting the growth of bacteria on surfaces. The method comprises thestep of contacting the surface with an effective amount of a compound ofthe present invention.

10. The compounds of the present invention have longer half-lives forNO-release than most known NO-releasing compounds. Thus, the compoundsof the present invention can be remain at internal treatment sites forlonger durations when used as a coating for implantable medical devicesand can be stored for longer periods of time than most knownNO-releasing compounds. They can also be used as bacteriostatic agents.

DETAILED DESCRIPTION OF THE INVENTION

11. The NO-releasing compounds of the present invention are smallorganic molecules. Thus, the compounds of the present invention arecomprised primarily of carbon and hydrogen, but can also include othernon-metallic elements such as sulfur, oxygen, nitrogen and/or halogens.The compounds of the present invention can contain functional groupswhich do not substantially increase the rate of NO release, for example,double the rate of release compared with the corresponding compoundwithout the functional group. Examples of suitable functional groupsinclude alcohols, thiols, amides, thioamides, carboxylic acids,aldehydes, ketones, halogens, double bonds, triples bonds and arylgroups (e.g, phenyl, naphthyl, furanyl, thienyl and the like).

12. As used herein, the term “small organic molecule” excludesmacromolecules such as a polypeptides, proteins, or S-nitrosylatedpolysaccharides or polymers, such as those disclosed in co-pending U.S.Ser. No. 08/691,862. The entire teachings of U.S. Ser. No. 08/691,862are incorporated herein by reference. The invention also excludesS-nitrosylated derivatives of the compounds represented by StructuralFormula (I)-(VII):

13. Each Ra is —H or methyl and is independently chosen.

14. As used herein, a “compound with a stabilized S-nitrosyl group”comprises, along with the S-nitrosyl group, a free thiol group or freealcohol group and has a half-life for NO release which is significantlygreater than for the corresponding compound with no free thiol oralcohol group (e.g., at least two times greater, and often about tentimes greater) when the same nitrosylating agent has been used toprepare both compounds. For example, the half-life for NO release for6-S-nitrosyl-hexane-1-thiol is about 1800 hours when prepared with SNAP(Example 2), whereas the half-life for NO release for1-S-nitrosyl-hexane is just over 200 hours when prepared with SNAP.Thus, 6-S-nitrosyl-hexane-1-thiol has a stabilized S-nitrosyl group.Generally, a compound with a stabilized S-nitrosyl group has a half lifefor NO release greater than about two hundred hours when nitrosylatedwith between about 0.5 to about 0.7 equivalents of SNAP, and oftengreater than about one thousand hours.

15. At least one —S-nitrosyl group in the disclosed compounds isstabilized by the interaction between a free thiol or a free alcoholgroup and the —S-nitrosyl group. Although Applicants do not wish to bebound by any particular mechanism, this stabilization is consistent withthe —S-nitrosyl group and free thiol (or alcohol) existing inequilibrium with a cyclic structure, as shown below in StructuralFormula (VIII):

16. —Y— is —O— or —S—. The “dashed” lines in Structural Formula (VIII)represent a stabilizing interaction, for example, a partial bond between—Y— and the sulfur atom and between —Y— and the nitrogen atom. Astabilizing interaction can be formed, for example, when a free thiol oralcohol is located within three covalent bonds of (alpha to) anS-nitrosyl group. In another example, a stabilizing interaction can beformed when a free thiol or alcohol can be brought within about one toone and a half bond lengths of an S-nitrosyl group by energeticallyaccessible conformational rotations of covalent bonds within themolecule.

17. Based on the discussion in the previous paragraph, the compounds ofthe present invention can also be described as comprising one or more ofthe cyclic structure shown in Structural Formula (VIII). The compoundsof the present invention can therefore also be represented by StructuralFormula (IX):

18. —Y— is —S— or —O—.

19. R is an organic radical. The term “organic radical”, as it is usedherein, refers to a moiety which comprises primarily hydrogen andcarbon, but can also include small amounts of other non-metallicelements such as sulfur, nitrogen, oxygen and halogens. R, when takentogether with the one or more stabilized S-nitrosyl groups or the one ormore cyclic structures depicted in Strutural Formula (IX), forms a smallorganic molecule, as described above.

20. n in Structural Formula (IX) is an integer, preferably from one toabout five. When n is greater than 1, the stabilized NO-releasingcompound has more than one stabilized —S-nitrosyl group. Each—S-nitrosyl group in a molecule requires a separate free thiol orseparate free alcohol for stabilization.

21. The compounds of the present invention preferably have a molecularweight less than about 1000 atomic mass units (hereinafter “amu”). Whenthe S-nitrosyl group is stabilized by an alcohol group, the compoundpreferably has a molecular weight greater than about 225 amu and morepreferably greater than about 500 amu. When the S-nitrosyl group isstabilized by a thiol group, the compound preferably has a molecularweight greater than about 375 amu, more preferably greater than about500 amu. When the S-nitrosyl group is stabilized by a thiol group andthe compound has a half-life for NO release of greater than about twohundred hours, the compound preferably has a molecular weight greaterthan about 225 amu.

22. A polythiol is a small organic molecule which has two or more freethiol groups. Preferably, a polythiol has between about two and aboutten free thiol groups.

23. A thioalcohol is a small organic molecule which has at least onealcohol group and at least one free thiol group. Preferably, athioalcohol has one to about five free thiol and one to about five freealcohol groups.

24. As used herein, the terms “polythiol” and “thioalocohol” do notinclude polypeptides or polythiolated polysaccharides and polymers withpendant thiol groups as described in co-pending, U.S. Ser. No.08/691,862. The terms “polythiol” and “thioalcohol” also specificallyexclude compounds represented by Structural Formulas (I)-(VII).

25. Suitable nitrosylating agents are disclosed in Feelisch and Stamler,“Donors of Nitrogen Oxides”, Methods in Nitric Oxide Research edited byFeelisch and Stamler, (John Wiley & Sons) (1996), the entire teachingsof which are hereby incorporated into this application by reference.Suitable nitrosylating agents include acidic nitrite, nitrosyl chloride,compounds comprising an S-nitroso group(S-nitroso-N-acetyl-D,L-penicillamine (SNAP), S-nitrosoglutathione(SNOG), N-acetyl-S-nitrosopenicillaminyl-S-nitrosopenicillamine,S-nitrosocysteine, S-nitrosothioglycerol, S-nitrosodithiothreitol andS-nitrosomercaptoethanol), an organic nitrite (e.g. ethyl nitrite,isobutyl nitrite, and amyl nitrite) peroxynitrites, nitrosonium salts(e.g. nitrosyl hydrogen sulfate), oxadiazoles (e.g.4-phenyl-3-furoxancarbonitrile) and the like. The half-life forNO-release of stabilized S-nitrosylated compounds can depend, at leastin part, on the nitrosylating agent used in their preparation. Forexample, the half-life for NO release of stabilized S-nitrosylatedcompounds prepared with SNAP is generally greater than the correspondingcompound prepared with tert-butyl nitrite (Example 2).

26. To prepare the compounds of the present invention, a polythiol or athioalcohol is reacted with between about 0.5 to about 0.7 equivalentsof nitrosylating agent with respect to each alcohol and thiol.Preferably, nitrosylating agent is added to the polythiol orthioalcohol. For example, to prepare a compound with a stabilizedS-nitrosyl group from 1,2-dithioethane or thioethanol, 1.0 mole ofnitrosylating agent is added to 1.0 mole of 1,2-dithioethane orthioethanol. S-nitroso-N-acetyl-D,L-penicillamine (SNAP) is a preferrednitrosylating agent. Larger amounts of nitrosylating agent with respectto thiol and/or alcohol groups can be used can be used with certainnitrosylating agents such as S-nitroso-N-acetyl-D,L-penicillamine(SNAP).

27. The nitrosylation reaction can be carried out neat or in a solventin which the polythiol or thioalcohol and the nitrosylating agent aresoluble. Commonly used solvents include dimethyl sulfoxide (DMSO),dimethyl formamide (DMF) and acetonitrile. Reaction temperatures betweenabout 0° C. and about 50° C., preferably ambient temperature, can beused. Concentrations of polythiol or thioalcohol are generally greaterthan about 0.01 M. Specific conditions for nitrosylation with SNAP andtert-butylnitrite (TBN) are provided in Example 1.

28. The reaction with acidic nitrite as the nitrosylating agent can be,for example, carried out in an aqueous solution with a nitrite salt,e.g. NaNO₂, KNO₂, LiNO₂ and the like, in the presence of an acid, e.g.HCl, acetic acid, H₃PO₄ and the like, at a temperature from about −20°C. to about 50° C., preferably at ambient temperature.

29. The reaction with NOCl as the nitrosylating agent can be carriedout, for example, in an aprotic polar solvent such as dimethylformamideor dimethylsulfoxide at a temperature from about −20° C. to about 50°C., preferably at ambient temperature.

30. The formation of stabilized S-nitrosyl groups is disfavored when thepolythiol or thioalcohol has substituents, particularly bulkysubstituents, in close proximity to the stabilizing thiol or alcoholgroups. For example, the formation of stabilized S-nitrosyl groups isdisfavored when three substituents, for example three alkyl groups, areattached to the carbon atoms alpha or beta to the thiol. AlthoughApplicants do not wish to be bound by any particular mechanism, it isbelieved that bulky groups in close proximity to a thiol or alcohol cansterically block the interaction between the thiol or alcohol and thethe S-nitrosyl group. Thus, —Y— and —S— in stabilized S-nitrosyl groupsare preferably bonded to methylene groups.

31. In a preferred embodiment, the compound is formed by nitrosylatingan esterified polyol represented by Structural Formula (X):

32. R is an organic radical, as described above.

33. n in Structural Formula (X) is an integer greater than two,preferably an integer from three to about ten. More preferably, n is aninteger from three to about eight.

34. Each X is independently a thiol-bearing aliphatic group or asubstituted thiol-bearing aliphatic group. Preferably, each X is thesame thiol-bearing aliphatic group. Examples of suitable thiol-bearingaliphatic groups include —CH₂SH, —CH₂CH₂SH, —CH₂CH₂CH₂SH and—CH₂CH₂CH₂CH₂SH.

35. The nitrosylation of the esterified polyol is carried out byreacting the esterified polyol with a nitrosylating agent, as describedabove, preferably with about 0.5 to about 0.7 equivalents ofnitrosylating agent per free thiol and free alcohol.

36. Compounds prepared by nitrosylating an esterified polyol representedby Structural Formula (X) have one or more stabilized S-nitrosyl groups.The compound formed by this reaction is represented by StructuralFormula (XI):

37. R is an organic radical, as described above.

38. Each X′ is an independently chosen aliphatic group or substitutedaliphatic group. Preferably each X′ is the same and is a C2-C6 alkylgroup, more preferably —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂— or —CH₂CH₂CH₂CH₂—.

39. p and m are positive integers such that p+m is greater than two.Preferably, p+m is less than or equal to about 10. Even more preferably,p+m are less than or equal to six.

40. Specific examples of polythiols and thioalcohols which have beennitrosylated to form compounds with stable S-nitrosyl groups are shownin the Table in Example 2. Also shown in the Table are the half-life forNO-release for each S-nitrosylated compound and the nitrosylating agentused to prepare each compound.

41. Compounds represented by Structural Formula (XI) can form polymers,which can be used to coat medical devices for delivering NO in vivo.These polymers are disclosed in co-pending U.S. Patent Application“NOVEL POLYMERS FOR DELIVERING NO IN VIVO” (Attorney Docket No.DUK96-08A), filed on Jun. 23, 1998, the entire teachings of which areincorporated herein by reference.

42. As used herein, aliphatic groups include straight chained, branchedor cyclic C₁- C₈ hydrocarbons which are completely saturated or whichcontain one or more units of unsaturation. Suitable substituents for analiphatic group are those which: 1) are substantially inert with respectto —S-nitrosyl groups, i.e., groups which do not substantially increasethe rate, e.g., double the rate of NO release from NO-releasingmolecules; and 2) do not substantially interfere with the nitrosylationof free thiol groups, i.e. do not substantially decrease the yield ofthe nitrosylation or cause the formation of significant amounts ofby-products. Examples of suitable substituents include halogens, C1-C5straight or branched chain alkyl groups, alcohols, carboxylic acids,amides, thioamides, and the like.

43. Another embodiment of the present invention is a method ofdelivering NO to a treatment site in a subject using the novelcompositions of the present inventions to deliver NO. A “treatment site”includes a site in the body of a subject in which a desirabletherapeutic effect can be achieved by contacting the site with NO. A“subject” refers to a human or an animal such as a veterinary animal(e.g., dogs, cats and the like) and farm animals (e.g., horses, cows,pigs and the like).

44. Treatment sites are found, for example, at sites within the bodywhich develop restenosis, injury or thrombosis as a result of traumacaused by contacting the site with a synthetic material or a medicaldevice. For example, restenosis can develop in blood vessels which haveundergone coronary procedures or peripheral procedures with PTCA ballooncatheters (e.g. percutaneous transluminal angioplasty). Restenosis isthe development of scar tissue from about three to six months after theprocedure and results in narrowing of the blood vessel. NO reducesrestenosis by inhibiting platelet deposition and smooth muscleproliferation. NO also inhibits thrombosis by inhibiting platelets andcan limit injury by serving as an anti-inflammatory agent.

45. A site in need of treatment with NO often develops at vascular siteswhich are in contact with a synthetic material or a medical device. Forexample, stents are often inserted into blood vessels to preventrestenosis and re-narrowing of a blood vessel after a procedure such asangioplasty. Platelet aggregation resulting in thrombus formation is acomplication which may result from the insertion of stents. NO is anantiplatelet agent and can consequently be used to lessen the risk ofthrombus formation associated with the use of these medical devices.Other examples of medical devices which contact vascular sites andthereby increase the risk of thrombus formation include sheaths forveins and arteries and GORE-TEX surgical prostheses.

46. The need for treatment with NO can also develop at non-vascularsites, for example at sites where a useful therapeutic effect can beachieved by reducing an inflammatory response. Examples include theairway, the gastrointestinal tract, bladder, uterine and corpuscavernosum. Thus, the compositions, methods and devices of the presentinvention can be used to treat respiratory disorders, gastrointestinaldisorders, urological dysfunction, impotence, uterine dysfunction andpremature labor. NO delivery at a treatment site can also result insmooth muscle relaxation to facilitate insertion of a medical device,for example in procedures such as bronchoscopy, endoscopy, laparoscopyand cystoscopy. Delivery of NO can also be used to prevent cerebralvasospasms post hemorrhage and to treat bladder irritability, urethralstrictures and biliary spasms.

47. The need for treatment with NO can also arise external to the bodyin medical devices used to treat bodily fluids temporarily removed frombody for treatment, for example blood. Examples include conduit tubeswithin heart lung machines, tubes of a dialysis apparatus and catheters.

48. The method of delivering NO to a treatment site in a subjectcontains implanting a medical device which comprises one or morecompounds of the present invention at the treatment site. NO can bedelivered to bodily fluids, for example blood, by contacting the bodilyfluid with a tube or catheter comprising one or more compounds of thepresent invention. Examples of treatment sites in a subject, medicaldevices suitable for implementation at the treatment sites and medicaldevices suitable for contacting bodily fluids such as blood aredescribed in the paragraphs hereinabove.

49. “Implanting a medical device at a treatment site” refers to bringingthe medical device into actual physical contact with the treatment siteor, in the alternative, bringing the medical device into close enoughproximity to the treatment site so that NO released from the medicaldevice comes into physical contact with the treatment site. A bodilyfluid is contacted with a medical device, e.g,., a tube or cather, whichcomprises one or more compounds of the present invention when, forexample, the bodily fluid is temporarily removed from the body fortreatment by the medical device, and the coating is an interface betweenthe bodily fluid and the medical device. Examples include the removal ofblood for dialysis or by heart lung machines.

50. An article, for example a medical device such as a stent, tube orcatheter, can be coated with one or more compounds of the presentinvention. A mixture is formed by combining a solution comprising adithiol or thioalcohol with an article insoluble in the solution. Themixture is then combined with a nitrosylating agent under conditionssuitable for nitrosylating free thiol groups, resulting in formation ofa stabilized NO-releasing compound. When the stabilized NO-releasingcompound is insoluble in solution, the NO-releasing compoundprecipitates from the solution and coats the article. When thestabilized NO-releasing compound is soluble in the solution or when thenitrosylation reaction is carried out in a polar aprctic solvent such asdimethylformamide (DMF) or dimethylsulfoxide (DMSO), the article can bedipped into or sprayed with the reaction mixture and then dried in vacuoor under a stream of an inert gas such as nitrogen or argon, therebycoating the article. Suitable nitrosylating agents include SNAP,tert-butyl nitrite, acidified nitrite, S-nitrosothiols, organic nitrite,nitrosyl chloride, oxadiazoles, nitroprusside and other metal nitrosylcomplexes, peroxynitrites, nitrosonium salts (e.g. nitrosylhydrogensulfate) and the like.

51. It is to be understood that other methods of applying coatings todevices, including methods known in the art, can be used to coatarticles with the compounds of the present invention.

52. An article incorporates an NO-releasing compound of the presentinvention when the compound is “entrapped” within the molecularframework of a material which is part of the article. For example, manymedical devices include certain polymers. An NO-releasing compound canbe incorporated into these polymers by carrying out the polymerizationreaction through which these polymers are formed in the presence of anNO-releasing compound. The NO-releasing compound is thereby entrapped inthe molecular framework of the resulting polymer product, which can thenbe used to prepare the medical device.

53. An article also incorporates an NO-releasing compound when theNO-releasing compound is chemically bonded to a material which is partof the article.

54. It is to be understood that other methods of incorporating compoundsinto compositions are known in the art and can be used to incorporatethe compounds of the present invention into the materials used toproduce medical devices.

55. The NO-releasing compounds of the present invention arebacteriostatic (Example 3). Thus, these compounds can be used to inhibitthe growth of bacteria on surfaces, for example, the surfaces of medicaldevices or medical furniture prior to use. “Inhibiting the growth ofbacteria” refers to a statistically significant lower bacteria count ona surface after application of the compound compared with a similarsurface which has not been treated with the compound. The NO-releasingcompound is applied to a surface in need of sterilization by, forexample, dissolving the compound in a non-toxic solvent atconcentrations between about 0.01 M and 5.0 M. The solution is thenapplied to the surface by spraying, wiping or pouring the resultingsolution onto said surface. A quantity of solution sufficient to coverthe surface is generally used. The solution is allowed to remain incontact with the surface for as long as inhibition of bacteria growth isrequired. The solution and any residues are removed, for example, bywiping or washing with a solvent suitable for dissolving theNO-releasing compound and any decomposition products.

56. The invention is further illustrated by the following examples,which are not intended to be limiting in any way.

EXEMPLIFICATION

57. All precursor thiols were obtained from Sigma-Aldrich Chemical Co.and were used without further purification. Tertiary-butyl nitrite (TBN,96%) and N-acetyl-D,L-penicillamine were purchased from Aldrich ChemicalCo. and were used without further purification. Dimethylsulfoxide (DMSO)was purchased from Mallinckrodt, passed through a CHELEX 100 column andbubbled for thirty minutes with argon prior to use. Dimethylformamide(DMF) was purchased from VWR Scientific.

58. S-Nitrosyl-N-acetyl-D,L-penicillamine (SNAP) was prepared by mixinga 0.2 M solution of N-acetyl-D,L-penicillamine in 1:1 methanol/1 N HClwith an equimolar amount of sodium nitrite in water. SNAP precipitatedout of solution as a green solid, which was filtered and washed withwater prior to use.

EXAMPLE 1 Preparation of Stabilized S-Nitrosylated Compounds

59. S-nitrosylated compounds were prepared by adding TBN to a polythiolor thioalcohol. Alternatively, S-nitrosylated compounds were prepared byadding a polythiol or thioalcohol to a solution of SNAP in DMF (0.12 Mis a representative concentration). All reactions were carried out atroom temperature. 0.5 equivalents of nitrosylating agent per thiol group(or per alcohol and thiol group) were used. All reactions were carriedout under argon in the dark. A rapid color change to red was observedafter addition of the nitrosylating agent. Stirring was continued forapproximately another two to five minutes after the color change.

60. S-nitrosylated compounds were characterized by ¹⁵N NMR and by theirUV/visible absorbance spectra. The ¹⁵N NMR spectrum of stabilizedS-nitrosylated compounds showed a singlet at about 424 ppm relative toHNO₃. In contrast, the ¹⁵N NMR spectrum of S-nitrosylated tertiary butylthiol prepared according to the procedure described above gave a singletat 480 ppm relative to HNO₃. The UV/visible absorbance spectrum ofstabilized S-nitrosylated compounds gave an absorbance maximum betweenabout 540 and 555 nanometers.

EXAMPLE 2 Half-Lives for NO Release From Stabilized S-NitrosylatedCompounds

61. The half-lives for NO release from the stabilized S-nitrosylatedcompounds of the present invention were determined by monitoring thedecrease in intensity of the absorbance maximum between 540 and 555nanometers over time. When the nitrosylation reaction was performedneat, the uv/visible absorbance spectrum was obtained by adding severaldrops of the reaction mixture to a cuvette containing DMSO. When thenitrosylation was carried out in solution, the absorbance spectrum wasobtained directly from the reaction mixture. Data for these plots wereobtained by performing at least three absorbance scans to create alinear plot from which half-lives were determined. Kinetics runs wereperformed in the dark under an argon atmosphere. A Hewlett-Packard 8452ADiode Array Spectrophotometer in conjunction with HP89531A MS-DOS UV/VISOperating Software was utilized to obtain kinetic data for use inhalf-life determinations.

62. The Table shows the half-lives for NO release of a number ofstablized S-nitrosylated polythiols and thioalcohols prepared accordingto the procedures described in Example 1. The Table indicates whetherthe half-life determination was for an S-nitrosylated compound preparedwith SNAP or TBN. Only approximate values could be determined when thehalf-life was greater than one thousand hours.

63. The Table also shows that the half-live for NO release forS-nitrosylated hexane thiol prepared with SNAP according to theprocedure described above is 280 hours and that the half-life for NOrelease of 1-S-nitrosyl hexane 6-thiol prepared with SNAP is about 1800hours. This result demonstrates the stabilizing effect of the free thiolgroup.

64. As can be seen from the Table, longer half-lives are generallyobtained when SNAP was used as the nitrosylating agent. S-Nitrosylatedcompounds obtained from SNAP in the Table generally have half-livesgreater than two hundred hours. TABLE Half- Life For NO- Release inHours: Compound TBN¹ SNAP² 104  225 87  570 84  618 108 126  440 167 170 196 1500³ 105  228 82  618 386 2200³ 33  150 48.8 1300³ 207.2 1800³ 188  280

EXAMPLE 3 Nitrosylated 3-Mercapto-1,2-Propanediol Is Bacteriostatic

65. Nitrosated 3-mercapto-1,2-propanediol was prepared with TBN bed inthe Example 1. The nitrosylated compound was dissolved in 10 mL ofdeionized water (0.16 M) that had been passed through a CHELEX 100column and bubbled with argon for 30 minutes. The resulting solution wasred.

66.E. coli cells (pTC 190) were plated onto the culture plates and grownovernight at 40° C. The E. coli cells contained a plasmid encoding forampicillin resistance. The solution was then sprayed onto a cultureplate (LB/Amp/Glucose). After 24 hours, this plate showed the growth ofa few colonies, all localized on one side of the plate.

67. A growth plate coated only with E. coli cells displayed a continuouslawn of growth after 24 hours. This plate was then sprayed with thenitrosylated compound solution at the 24^(th) hour. After another 24hours the plate contained a lawn of colonies that was approximately asdense as prior to application of the S-nitrosylated compound.

68. A control plate that was not coated with the nitrosylated compoundsolution nor plated with E. coli cells showed no bacterial growth. Asecond control plate that was not plated with E. coli cells but coatedwith the nitrosylated compound solution showed no bacterial colonygrowth.

69. These results show that nitrosylated 3-mercapto-1,2-propanediol isbacteriostatic.

EXAMPLE 4 Reaction of 1,6-Hexanedithiol and 1-Hexanethiol With SNAP

70. 1,6-Hexanedithiol (25 uL, 24.6 mg, 0.164 mmol) was dissolved in 6.0mL of DMSO in 11 separate 10-mL test tubes.S-Nitroso-N-acetylpenicillamine (SNAP) was added in incrementalstoichiometric amounts to each test tube as follows: mg SNAP equivalentsSNAP 9.0 0.25 18.0 0.5 27.0 0.75 36.0 1.0 45.0 1.25 54.0 1.5 63.0 1.7572.0 2.0 108.0 3.0 144.0 4.0 180.0 5.0

71. The samples were stored under ambient atmosphere in the dark.Absorbance values were taken at time intervals in a 1-cm path lengthquartz cell at wavelengths of 520, 554, and 594 nm. While the absorbancereadings were being taken, the samples were under ambient light forapproximately 1 hour.

72. 1-Hexanethiol (25 uL, 21.0 mg, 0.177 mmol) was dissolved in 6.0 mLof DMSO in 9 separate 10-mL test tubes. S-Nitroso-N-acetylpenicillamine(SNAP) was added in incremental stoichiometric amounts to each test tubeas follows: mg SNAP equivalents SNAP 9.8 0.25 19.5 0.5 29.3 0.75 39.01.0 48.8 1.25 58.5 1.5 78.0 2.0 117.0 3.0 156.1 4.0

73. The samples were stored under ambient atmosphere in the dark.Absorbance values were taken at time intervals in a 1-cm path lengthquartz cell at wavelengths of 520, 554, and 594 nm. While the absorbancereadings were being taken, the samples were under ambient light forapproximately 1 h.

74. In both reactions, a red color corresponding to an absorbance at 554nm appeared almost immediately after the reagents are mixed. The redcolor peaked at the same maximum value about three hours after mixingfor both 1-hexanethiol and 1,6-hexanedithiol when 0.5 equivalents ormore of SNAP are used. This result is consistent with the formation of astabilized S-nitrosylated compound, i.e., the nitrosylation of only oneof the thiol groups in 1,6-hexanedithiol, even when more than 0.5equivalents of SNAP are present.

75. When the experiment was repeated with TBN or acidic nitrite in placeof SNAP, the absorbance peak at 554 nm for the reaction with1,6-hexanedithiol increased as the amount of nitrosylating usedincreased, up to 1.0 equivalent of nitrosylating agent per thiol group.This result is consistent with the formation of stabilizedS-nitrosylated when 0.5 equivalents of TBN or acidic nitrite were used,and with nitrosylation of the second thiols group when more than 0.5equivalents of TBN or acidic nitrite were used.

EQUIVALENTS

76. While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

What is claimed:
 1. An NO-releasing compound comprising at least onestabilized S-nitrosyl group and at least one free thiol group or freealcohol group, wherein the S-nitrosyl is stabilized by the free alcoholgroup or the free thiol group, with the proviso that the compound is nota polypeptide, an S-nitrosylated polysaccharide or a polymer withpendant S-nitrosyl groups and is not an S-nitrosylated derivative of acompound represented by one of the following structural formulas:

wherein each Ra is —H or methyl and is independently chosen.
 2. Thecompound of claim 1 wherein the compound has at least one free alcoholgroup and has a molecular weight greater than about 225 atomic massunits.
 3. The compound of claim 2 wherein the compound has a molecularweight less than about 1000 atomic mass units.
 4. The compound of claim3 wherein the free alcohol group(s) and the S-nitrosyl group(s) are eachbonded to a methylene group.
 5. The compound of claim 3 wherein thecompound has a half-life for NO release of greater than about twohundred hours.
 6. The compound of claim 1 wherein the compound has atleast one free thiol group and has a molecular weight greater than about375 atomic mass units.
 7. The compound of claim 6 wherein the compoundhas a molecular weight less than about 1000 atomic mass units.
 8. Thecompound of claim 1 wherein the compound has at least one free thiolgroup, a molecular weight greater than about 225 atomic mass units and ahalf-life for NO release greater than about two hundred hours.
 9. Thecompound of claim 6 wherein the free thiol group(s) and the S-nitrosylgroup(s) are each bonded to a methylene group.
 10. The compound of claim1 wherein the compound is represented by the following structuralformula:

wherein: R is an organic radical; each X′ is independently a substitutedor unsubstituted aliphatic group; and p and m are each a positiveinteger such that p+m is greater than two.
 11. The compound of claim 10wherein every X′ is the same.
 12. The compound of claim 11 wherein p+mis less than or equal to about ten.
 13. The compound of claim 12 whereineach X′ is —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂— or —CH₂CH₂CH₂CH₂—.
 14. Acompound prepared by reacting a polythiol or a thioalcohol with anitrosylating agent, with the proviso that the polythiol or thioalcoholis not a polypeptide, a polythiolated alcohol, or a polymer with pendantthiol groups and is not represented by one of the following structuralformulas:

wherein each Ra is —H or methyl and is independently chosen.
 15. Thecompound of claim 14 wherein about 0.5 to about 0.7 equivalents ofnitrosylating agent per free thiol and per free alochol in the polythiolor thialcohol is used.
 16. The compound of claim 15 wherein the compoundis prepared by reacting a polythiol with a nitrosylating agent, whereinthe polythiol is an esterified polyol represented by the followingstructural formula:

wherein: R is an organic radical; n is a positive integer greater thantwo; and each X is independently a substituted or unsubstitutedthiol-bearing aliphatic group.
 17. The compound of claim 16 wherein eachX is the same and n is less than about
 10. 18. The compound of claim 17wherein each X is —CH₂SH, —CH₂CH₂SH, —CH₂CH₂CH₂SH or —CH₂CH₂CH₂CH₂SH.19. The compound of claim 15 wherein the nitrosylating agent is reactedwith a thioalcohol and the compound has a molecular weight of at leastabout 225 atomic mass units.
 20. The compound of claim 19 wherein thecompound has a molecular weight less than about 1000 atomic mass units.21. The compound of claim 20 wherein the alcohol and thiol are bothprimary.
 22. The compound of claim 15 wherein the nitrosylating agent isreacted with a polythiol and the compound has a molecular weight greaterthan about 375 atomic mass units.
 23. The compound of claim 22 whereinthe compound has a molecular less than about 1000 atomic mass units. 24.The compound of claim 23 wherein both thiols are primary.
 25. Thecompound of claim 14 wherein the nitrosylating agent isS-nitroso-N-acetyl-D,L-penicillamine.
 26. A method of preparing anNO-releasing compound with a stabilized S-nitrosyl group, said methodcomprising the step of reacting a polythiol or a thioalcohol with anitrosylating agent, with the proviso that the polythiol or thioalcoholis not a polypeptide, a polythiolated alcohol, or a polymer with pendantthiol groups and is not represented by one of the following structuralformulas:

wherein each Ra is —H or methyl and is independently chosen.
 27. Themethod of claim 26 wherein about 0.5 to about 0.7 equivalents ofnitrosylating agent per free thiol and per free alcohol in the polythiolor thialcohol is used.
 28. The method of claim 27 wherein the compoundis prepared by reacting a polythiol with a nitrosylating agent, whereinthe polythiol is an esterified polyol represented by the followingstructural formula:

wherein: R is an organic radical; n is a positive integer greater thantwo; and each X is independently a substituted or unsubstitutedthiol-bearing aliphatic group.
 29. The method of claim 28 wherein each Xis the same and n is less than about
 10. 30. The method of claim 29wherein each X is —CH₂SH, —CH₂CH₂SH, —CH₂CH₂CH₂SH or —CH₂CH₂CH₂CH₂SH.31. The method of claim 27 wherein the nitrosylating agent is reactedwith a thioalcohol and the compound has a molecular weight of at leastabout 225 atomic mass units.
 32. The method of claim 31 wherein thecompound has a molecular weight less than about 1000 atomic mass units.33. The method of claim 33 wherein the alcohol and thiol are bothprimary.
 34. The method of claim 27 wherein the nitrosylating agent isreacted with a polythiol and the compound has a molecular weight greaterthan about 375 atomic mass units.
 35. The method of claim 34 wherein thecompound has a molecular less than about 1000 atomic mass units.
 36. Themethod of claim 35 wherein both thiols are primary.
 37. The method ofclaim 26 wherein the nitrosylating agent isS-nitroso-N-acetyl-D,L-penicillamine.
 38. An article capable ofreleasing NO wherein the article contains the NO-releasing compound ofclaim 1 .
 39. The article of claim 38 wherein the article is a medicaldevice suitable for implantation at a treatment site in a subject. 40.The article of claim 38 wherein the article is a medical device used totemporarily remove a bodily fluid from a subject.
 41. The article ofclaim 40 wherein the medical device is a tube of catheter.
 42. Thearticle of claim 38 wherein the NO-releasing compound coats the surfacesof the article.
 43. An article capable of releasing NO wherein thearticle contains the NO-releasing compound of claim 14 .
 44. The articleof claim 43 wherein the article is a medical device suitable forimplantation at a treatment site in a subject.
 45. The article of claim43 wherein the article is a medical device used to temporarily remove abodily fluid from a subject.
 46. The article of claim 45 wherein themedical device is a tube of catheter.
 47. The article of claim 43wherein the NO-releasing compound coats the surfaces of the article. 48.A method of delivering nitric oxide to a treatment site in a subject,comprising the step of implanting a medical device at the treatmentsite, wherein: a) the medical device contains the NO-releasing of claim1 ; and b) the medical device is suitable for implantation at thetreatment site in the subject.
 49. A method of delivering nitric oxideto a bodily fluid of a subject, comprising the step of contacting thebodily fluid with a medical device which contains the NO-releasingcompound of claim 1 .
 50. The method of claim 49 wherein theNO-releasing compound coats the surfaces of the medical device.
 51. Amethod of delivering nitric oxide to a treatment site in a subject,comprising the step of implanting a medical device at the treatmentsite, wherein: a) the medical device contains the NO-releasing of claim14 ; and b) the medical device is suitable for implantation at thetreatment site in the subject.
 52. A method of delivering nitric oxideto a bodily fluid of a subject, comprising the step of contacting thebodily fluid with a medical device which contains the NO-releasingcompound of claim 14 .
 53. The method of claim 52 wherein theNO-releasing compound coats the surfaces of the medical device.
 54. Amethod of sterilizing a surface comprising the step of contacting thesurface with an effective amount of the NO-releasing compound of claim14 .